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Osteoarthritis (OA), which is characterized by articular cartilage degeneration, shows a gradually increasing incidence with age. This study explored the molecular mechanism underlying the proliferation and apoptosis of chondrocytes during OA progression. In this study, chondrocytes were isolated from human knee cartilages. The targeted relationship among nuclear enriched abundant transcript 1 (NEAT1), microRNA-543 (miR-543) and PLA2G4A was predicted on TargetScan V7.2 and starBase and validated by performing dual-luciferase reporter assay. High-expressed NEAT1 was detected in OA cartilage and chondrocytes. NEAT1 was negatively correlated with miR-543 and was low-expressed in OA cartilage and PLA2G4A was negatively correlated with miR-543 and was high-expressed in OA cartilage. In OA chondrocytes, the overexpressed NEAT1 inhibited the expressions of p-Akt1 and Bcl-2 and upregulated that of matrix metalloprotease (MMP)-3, MMP-9, MMP-13, interleukin (IL)-6 and IL-8, but such effects of overexpressed NEAT1 were reversed by miR-543 mimic. SiRNA-NEAT1 exerted an opposite effect to NEAT1 overexpression on OA chondrocytes, but this could be reversed by miR-543 inhibitor. The effect of PLA2G4A overexpression was the opposite to miR-543 mimic on OA chondrocytes. In conclusion, NEAT1 could sponge miR-543 to induce PLA2G4A expression, inhibit chondrocyte proliferation and promote apoptosis.

A full-length cDNA sequence of plant type CRY (designated Hae-P-CRY) was cloned from the green alga Haematococcus pluvialis. The cDNA sequence was 3608 base pairs (bp) in length, which contained a 2988-bp open reading frame encoding 995 amino acids with molecular mass of 107.7 kDa and isoelectric point of 6.19. Multiple alignment analysis revealed that the deduced amino acid sequence of Hae-P-CRY shared high identity of 47-66% with corresponding plant type CRYs from other eukaryotes. The catalytic motifs of plant type CRYs were detected in the amino acid sequence of Hae-P-CRY including the typical PHR and CTE domains. Phylogenetic analysis showed that the Hae-P-CRY was grouped together with other plant type CRYs from green algae and higher plants, which distinguished from other distinct groups. The transcriptional level of Hae-P-CRY was strongly decreased after 0-4 h under HL stress. In addition, the Hae-P-CRY gene was heterologously expressed in Escherichia coli BL21 (DE3) and successfully purified. The typical spectroscopic characteristics of plant type CRYs were present in Hae-P-CRY indicated that it may be an active enzyme, which provided valuable clue for further functional investigation in the green alga H. pluvialis. These results lay the foundation for further function and interaction protein identification involved in CRYs mediated signal pathway under HL stress in H. pluvialis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Ammonia is toxic to most bony fishes. However, little information is available on the toxicology mechanisms induced by ammonia and the means to mitigate the effects by various fishes. In this study, four groups of experiments were designed and carried out to test the response of dolly varden char to ammonia toxicity and their mitigation through methionine sulfoximine (MSO). NaCl group was injected with NaCl, NH 3 group was injected with ammonium acetate, NH 3 +MSO group was injected with ammonium acetate and MSO, MSO group was injected with MSO. Results showed that ammonia toxicity could lead to blood deterioration (elevation in white blood cell and blood ammonia), free amino acid imbalance (elevation in glutamine, glutamate, arginine and ornithine, coupled with reduction of citrulline and aspartate), ammonia metabolism enzyme activity inhibition (reduction in carbamyl phosphate synthetase, ornithine transcarbamylase and arginase), oxidative stress (reduction in superoxide dismutase, catalase and glutathione peroxidase) and immunosuppression (reduction in lysozyme, 50% hemolytic complement, total immunoglobulin and phagocytic index), but the MSO can eliminate fatal effect of oxidative damage. In addition, ammonia poisoning could induce down-regulation of antioxidant enzymes coding genes (SOD, CAT and GPx) and up-regulation of inflammatory cytokine genes (TNFα, IL-1β and IL-8) transcription, suggesting that immunosuppression and inflammation may relate to oxidative stress in fish., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Ischemic stroke is the third leading cause of death. There is no known treatment or cure for the disease. Moreover, the pathological mechanism of ischemic stroke remains unclear., Objective: We aimed to identify potential microRNAs (miRNAs) and mRNAs, contributing to understanding the pathology of ischemic stroke., Methods: First, the data of miRNA and mRNA were downloaded for differential expression analysis. Then, the regulatory network between miRNA and mRNAs was constructed. Third, top 100 differentially expressed mRNAs were used to construct a protein-protein interaction network followed by the function annotation of mRNAs. In addition, in vitro experiment was used to validate the expression of mRNAs. Last, receiver operating characteristic diagnostic analysis of differentially methylated genes was performed., Results: Totally, up to 26 differentially expressed miRNAs and 1,345 differentially expressed mRNAs were identified. Several regulatory interaction pairs between miRNA and mRNAs were identified, such as hsa-miR-206-HMGCR/PICALM, hsa-miR-4491-TMEM97, hsa-miR-3622b-5p/hsa-miR-548k-KLF12, and hsa-miR-302a-3p/hsa-miR-3145-3p-CTSS. MAPK signaling pathway (involved DUSP1) and the Notch signaling pathway (involved NUMB and CREBBP) were identified. The expression validation of KLF12, ARG1, ITGAM, SIRT4, SERPINH1, and DUSP1 was consistent with the bioinformatics analysis. Interestingly, hsa-miR-206, hsa-miR-4491, hsa-miR-3622b-5p, hsa-miR-548k, hsa-miR-302a-3p, hsa-miR-3145-3p, KLF12, and ID3 had the potential diagnostic value of ischemic stroke., Conclusions: The identified differentially expressed miRNAs and mRNAs may be associated with the development of ischemic stroke., (© 2020 S. Karger AG, Basel.)

Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, β-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, β-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia., (© The Author(s) 2020. Published by Oxford University Press on behalf of Entomological Society of America.)

A 56-day growth trial was conducted to investigate the effects of dietary yeast hydrolysate on the growth performance, antioxidation, immune response and resistance against Aeromonas hydrophila in largemouth bass. Four experimental diets were prepared with yeast hydrolysate levels of 0% (Y0), 1.5% (Y1.5), 3.0% (Y3.0) and 4.5% (Y4.5). Each diet was randomly assigned to triplicate 150-L tanks and each tank was stocked with 30 largemouth bass (initial body weight, IBW = 7.71 ± 0.02 g). A challenge test was carried out after the feeding trial by injecting A. hydrophila intraperitoneally for 4-day observation. The results showed that the FBW and WGR in Y1.5 group were significantly higher than those in Y0 group (P < 0.05) and the feed conversion ratio (FCR) got the lowest value in Y1.5 group. And the hydrolysate supplement significantly increased the 4-day cumulative survival rate after the bacterial challenge (P < 0.05). The plasma malondialdehyde was lower in the yeast hydrolysate supplement groups in both pre- and post-challenge test (P < 0.05), while the plasma C3 increased (P < 0.05). In post-challenge test, the plasma superoxide dismutase (SOD) and catalase (CAT) activities increased in the Y1.5 and Y3.0 groups respectively (P < 0.05), and plasma lysozyme in Y1.5 group and the plasma IgM in Y3.0 group were higher than those in others respectively (P < 0.05). For the q-PCR results, in post-challenge test, the hepatic hep2 expression level in Y1.5 and Y4.5 groups were both significantly higher than those in others (P < 0.05), as well as il-8 in Y3.0 group. The spleen hif-1alpha and tgf-beta1 expression levels in Y4.5 group were all significantly lower than those in others (P < 0.05), while the gilt was significantly higher (P < 0.05) in the post-challenge test. And the expression levels of spleen tnf-alpah1 in Y1.5 and Y3.0 groups and il-8 in Y3.0 group were all significantly higher than those in other groups (P < 0.05) in the post-challenge test. The head kidney gilt expression level was significantly higher in the yeast hydrolysate supplement groups compared with the Y0 group (P < 0.05), and the head kidney il-8 expression level in Y1.5 group was significant higher than those in other groups in post-challenge test (P < 0.05). The present results indicated dietary yeast hydrolysate improved the antioxidant ability and enhanced the immune response of largemouth bass without negative effect on growth. And 1.5% or 3.0% of dietary yeast hydrolysate was recommended for largemouth bass based on the present results., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

β 2 -adrenergic receptor (β 2 -AR) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of β-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and 59.57 μg/l, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was 3.2 μg/l and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified β 2 -AR would have great application potential in detection of β-agonist residues.

It has been long demonstrated that cage rearing (CR) deprives the animal of the possibility to express natural behaviors and results in stress. However, the effect of the rearing system on gene expression and the molecular levels of the gut microbiome are unknown. 10-wk-old Beijing You chickens were studied in parallel CR and free-range (FR) systems for 30 wk, to investigate the effect of rearing systems on behavior, microbiota composition, and gene expression. From week 40, a match-mismatch design was conducted for 5 wk. The results indicated that CR deprives the animals of natural behaviors, evidenced by sham dust-bathing behavior. A decreased alpha diversity of gut microbiome composition of CR chickens was seen in FR compared to CR-FR chickens (P < 0.001), and the alpha diversity of gut microbiome composition of FR-CR was decreased as compared to FR chickens (P = 0.045). The heat map and beta-diversity analysis showed that the cluster of gut microbial compositions were similar between the mismatch groups (FR-CR and CR-FR), while those of CR showed the lowest diversity from the 4 groups. The relative abundance of gut microbes at genera and species levels was different between comparisons (P < 0.05). Moreover, the CR (both CR and FR-CR) triggered the downregulation of most Kyoto encyclopedia of genes and genomes pathways, while it was upregulated in 2 genetic information processing pathways, compared to FR hens regardless of long or short term. In conclusion, CR deprived chickens of their normal behavior and resulted in changes in the microbiome diversity and pathways and gene expression of chickens., (© 2019 Poultry Science Association Inc.)

Microglia play a pivotal role in maintaining homeostasis of the CNS. There is growing interest in understanding how microglia influence normal brain function and disease progression. Several microglia-specific Cx3cr1-Cre lines have been developed and have become indispensable tools in many investigations of microglial function. However, some recent studies have reported that these lines may have significant leakage into neurons. Other studies have reported that Cx3cr1 is expressed in non-microglial cells, including neurons and astrocytes, in vitro or in vivo either during brain development or upon neurological insult. All these reports raise serious concerns about the trustworthiness of these Cre-lines and whether the conclusions drawn from previous studies are valid. Here, we found that a floxed fluorescent reporter mouse line which has been frequently used to verify Cre lines displayed spontaneous expression of the GFP reporter, independent of Cre recombinase, thus revealing a potential caveat in assessing cre lines. We further confirmed that two Cx3cr1-Cre mouse lines can drive fluorescent reporter expression largely restrictively in microglia. Finally, we clarified that these two mouse lines maintain microglia-specific expression even following excitatory injury. Together, our findings confirm that two previously created Cx3cr1-Cre lines remain as invaluable tools for studying microglia. Moreover, to ensure the quality of data generated and the soundness of conclusions drawn from such data, it should be compulsory to thoroughly examine reporter lines for spontaneous leakiness when labeling cells to study CNS function and diseases., (Copyright © 2019 Zhao et al.)

Intracerebral hemorrhage (ICH) is a severe neurological disorder with no proven treatment. Our prior research identified a significant association with monocyte level and ICH mortality. To advance our understanding, we sought to identify gene expression after ICH using a swine model to test the hypothesis that ICH would induce peripheral blood mononuclear cell (PBMC) gene expression. In 10 pigs with ICH, two PBMC samples were drawn from each with the first immediately prior to ICH induction and the second six hours later. RNA-seq was performed with subsequent bioinformatics analysis using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity® Pathway Analysis (IPA). There were 182 significantly upregulated and 153 significantly down-regulated differentially expressed genes (DEGs) after ICH. Consistent with findings in humans, significant GO and KEGG pathways were primarily related to inflammation and the immune response. Five genes, all upregulated post-ICH and known to be associated with monocyte activation, were repeatedly DEGs in the significant KEGG pathways: CD14, TLR4, CXCL8, IL-18, and CXCL2. In IPA, the majority of upregulated disease/function categories were related to inflammation and immune cell activation. TNF and LPS were the most significantly activated upstream regulators, and ERK was the most highly connected node in the top network. ICH induced changes in PBMC gene expression within 6 h of onset related to inflammation, the immune response, and, more specifically, monocyte activation. Further research is needed to determine if these changes affect outcomes and may represent new therapeutic targets.

Background: This study was performed to determine the mRNA expression levels of limb-bud and heart (LBH) in peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis (RA), and to assess the associations of LBH rs7579944 and rs906868 polymorphisms with RA in a Chinese population. Methods: Real-time transcription-polymerase chain reaction analysis was used to determine the LBH mRNA expression in PBMCs from 53 patients with RA and 58 healthy controls, and LBH rs7579944 and rs906868 polymorphisms were genotyped in 328 RA patients and 449 healthy controls. Results: The LBH mRNA expression levels were significantly decreased in RA patients compared with healthy controls (p = 0.02). However, non-significant correlation between LBH mRNA expression in PBMCs and ESR (r = 0.14, p = 0.36), CRP (r = 0.17, p = 0.27) or DAS28-ESR (r = 0.23, p = 0.12) was found. There was no significant difference in neither genotype (p = 0.90) nor allele (p = 0.97) distribution of the LBH rs7579944 polymorphism between RA patients and healthy controls. The relationships between the major allele T of LBH rs7579944 polymorphism and the risk of RA under dominant and recessive model were examined, whereas non-significant evidence was found. Similarly, a non-significant signal was detected for the association of LBH rs906868 polymorphism with RA. Conclusions: Decreased expression of LBH mRNA in PBMCs might contribute to the pathogenesis of RA.

Arsenic trioxide (As2O3; ATO), a traditional Chinese medicine, is used to treat patients with acute promye-locytic leukemia, while its application for treatment of systemic lupus erythematosus (SLE) is still under evaluation. The high expression of INF-gamma (INF-γ) is a primary pathogenic factor in SLE. It is found that ATO can reduce INF-γ expression levels in lupus-prone mice, whereas it is not clear whether ATO has the same effect on SLE patients. Therefore, this study was to investigate the underlying mechanism of the effects of ATO on the expression of INF-γ in splenocytes of MRL/lpr mice and PBMCs of human lupus. The mRNA and protein expression levels of INF-γ were assessed by real-time RT-PCR and ELISA, respectively. The histone acetylation status of the INF-γ promoter and the binding of RNA polymerase II (RNA Pol II) to the INF-γ promoter were detected using a chromatin immunoprecipitation (ChIP) technique. The mRNA and protein expression levels of INF-γ decreased in both splenocytes of MRL/lpr mice and PBMCs of SLE patients with ATO treatment, which were accompanied by reduced histone H4 and H3 acetylation in INF-γ promoter and decreased combination of RNA Pol II to the INF-γ promoter. Therefore, ATO may reduce the expression level of the INF-γ by altering the levels of INF-γ promoter acetylation and the combination of RNA Pol II to the INF-γ promoter in splenocytes of MRL/lpr mice and PBMCs of SLE patients.

The pathogenesis and etiology of pancreatic cancer remain to be fully elucidated; therefore, associated investigations are required to improve the outcome and prognosis of patients. In the present study, the effects of the overexpression of p16ink4a on the Wnt/β‑catenin signaling pathway were investigated in pancreatic cancer cell lines. Two pancreatic cancer cell lines, Bxpc‑3 and Miapaca‑2, characterized by low expression of p16ink4a, were transfected with the pc‑DNA3.0‑p16ink4a plasmid. After 24 h, Reverse transcription‑polymerase chain reaction and western blot analyses were performed to evaluate the expression of p16ink4a, β‑catenin, which is a key molecule in the Wnt/β‑catenin signaling pathway, c‑myc and cyclin D1, which are molecules downstream of β‑catenin. The expression of p16ink4a was significantly upregulated in the transfected cells. Consequently, the expression of β‑catenin was inhibited, whereas the expression levels of c‑myc and cyclin D1 were not altered significantly. The increased expression of p16ink4a may affect the activity of Wnt/β‑catenin signaling through modulation of the expression of β‑catenin. The results of the present study provide information for the future development of targeted treatments for pancreatic cancer.

Epigenetic alteration plays critical roles in gliomagenesis by regulating gene expression through modifications of Histones and DNA. Trimethylation of H3K9, an essential repressed transcription mark, and one of its methyltransferase, SUV39H1, are implicated in glioma pathogenesis and progression. We find that the protein level of HP1α, a reader of H3K9me3 is elevated in GOS3 and 1321N1 glioma cell lines. H3K9me3 and SUV39H1 level are also upregulated. Depletion of HP1α and SUV39H1 weakens GOS3 and 1321N1 cell proliferation capacity and results in apoptosis of cells. Furthermore, we find that HP1α and H3K9me3 are enriched in the FAS and PUMA promoters, which suggests that upregulated HP1α and H3K9me3 prevent apoptosis by suppressing apoptotic activators. These data indicates that up-regulated HP1α, SUV39H1, and H3K9me3 in glioma cells are functionally associated with glioma pathogenesis and progression, and may serve as novel biomarkers for future diagnostic and therapeutic targeting of brain tumors.

Objective: While a prognosis value of progesterone receptor (PR) in ovarian cancer has been reported in some publications, controversial data were presented by different reports. In order to address the disagreement of progesterone receptor in ovarian cancer survival, we conducted this meta-analysis., Methods: Relevant articles on progesterone receptor and ovarian cancer prognosis were identified via a thorough search of PubMed, Embase and Cochrane Central. Hazard ratios (HR) and 95% confidence interval (CI) were extracted from studies on overall survival (OS) and disease-free survival (DFS)/progress-free survival (PFS)/recurrence-free survival (RFS)., Result: A total of 28 eligible studies containing 5685 patients were collected for analysis. It was found that progesterone receptor positivity was significantly associated with favorable overall survival (OS) (HR = 0.86, 95% CI = 0.78 to 0.95, P = 0.002) and disease-free survival (DFS)/progress-free survival (PFS)/recurrence-free survival (RFS) (HR = 0.75, 95% CI = 0.61 to 0.93, P = 0.008) of ovarian cancer patients. Subgroup analysis showed that progesterone receptor expression was associated with a favorable prognosis of unclassified ovarian cancer, European origin, and immunohistochemical detection method., Conclusion: Progesterone receptor expression can be used as a favorable prognostic predictor in ovarian cancer managements.

Human equilibrative nucleoside transporters (hENT) 1 and 2, encoded by SLC29A1 and SLC29A2, permit the bidirectional passage of nucleoside analogues into cells and may correlate with clinical responses to chemotherapy in patients with colorectal cancer (CRC). The purpose of this study was to evaluate the expression profiles of SLC29A1 and SLC29A2 in human cancer cell lines. Using quantitative real-time polymerase chain reaction, we comprehensively profiled the transcription levels of SLC29A1 and SLC29A2 in 16 colon cancer cell lines. We validated the ubiquitous and heterogeneous distribution of SLC29A1 and SLC29A2 in human colon cancer cell lines and demonstrated that SLC29A1 was highly expressed in 25% of metastatic cell lines (Colo201 and Colo205) and 62.5% of primary cell lines (Caco2, Colo320, HCT116, RKO, and SW48). For the first time, we showed that both SLC29A1 and SLC29A2 were expressed at lower levels in colon cancer cell lines originating from metastatic sites than from primary sites. These findings indicate that most patients with metastatic CRC (mCRC) may have low hENT1 expression, and treatment with nucleoside analogues may be inefficient. However, some patients still show high hENT1 expression and have a high probability of benefiting from these drugs. Therefore, evaluating transporter expression profiles and different drug responses between primary and metastatic tumors in patients with mCRC is important. Further assessment of the association between hENTs and drug-based treatment of mCRC is required to elucidate the mechanisms of chemotherapy resistance.

Distal-less homeobox 2 (Dlx2), a member of the Dlx family of transcription factors, is important for the development of craniofacial tissues. Previous studies based on knock‑out mutant mice revealed that Dlx2 primarily disturbed the development of tissues from maxillary arch. The present study used a transgenic mouse model to specifically overexpress Dlx2 in neural crest cells in order to investigate the role of Dlx2 overexpression in post‑natal condyle in mice. The model was constructed and the phenotype observed using gross observation, micro‑CT scan and histological examination. The model determined that overexpression of Dlx2 may lead to postnatal condyle malformation, subchondral bone degradation and irregular histological structure of the condylar cartilage. In addition, the expression of osteocalcin in the condyle region was markedly downregulated, whereas expression of msh homeobox 2 was upregulated. The results of the present study suggest that Dlx2 overexpression in cranial neural crest cells would disrupt the development of post‑natal condyle, which demonstrates that the expression level and the spatiotemporal expression patterns of Dlx2 may be important in regulating the development of post-natal condyle in mice, and also offered a possible temporal‑mandibular joint osteoarthritis model animal for future studies.

Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member of the IL-1 family, interleukin-38 (IL-38, IL-1F10, or IL-1HY2), which was discovered in 2001. Although the functional role of IL-38 remains unclear, recent reports show that IL-38 binds to the IL-36 receptor (IL-36R) which is also targeted by the IL-36 receptor antagonist (IL-36Ra). Consequently, these two molecules have similar effects on immune cells. Here, we describe the expression of soluble and active recombinant IL-38 in Escherichia coli (E. coli). The IL-38 gene sequence was optimized for expression in E. coli and then cloned into a pEHISTEV expression vector, which has an N-terminal 6-His affinity tag under control of the T7 lac strong promoter. Optimization of culture conditions allowed induction of the recombinant fusion protein with 0.1 mM isopropyl β-D-1-thio galactoside (IPTG) at 37°C for 4h. The recombinant fusion protein was purified using an Ni affinity column and was further digested with TEV protease; the cleaved protein was purified by molecular-exclusion chromatography. Next, we measured IL-38 binding ability using functional ELISA. The purified proteins were used to immunize a New Zealand white rabbit four times to enable the production of polyclonal antibodies. The specificity of the prepared polyclonal antibodies was determined using Western blot, and the results showed they have high specificity against IL-38. Here, we describe the development of an effective and reliable method to express and purify IL-38 and anti-IL-38 antibodies. This will enable the function and structure of IL-38 to be determined., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Parenteral nutrition in infants with gastrointestinal disorders can be lifesaving, but it is also associated with parenteral nutrition-associated liver disease. We investigated the effects of incorporating ω-3 fish oil in a parenteral nutrition mixture on signs of parenteral nutrition-associated liver disease and explored the mechanism involved in this process., Methods: Seven-day-old New Zealand rabbits were divided into 3 groups of 8, and for 1 week they were infused via the right jugular vein with standard total parenteral nutrition with soybean oil (TPN-soy) or TPN with ω-3 fish oil-based lipid emulsion (TPN-FO), or naturally nursed with rabbit milk (control). Serum and liver tissues were analyzed for serological indicators and pathology, respectively. Reverse-transcriptase polymerase chain reaction was used to evaluate the messenger RNA levels of the endoplasmic reticulum stress chaperone protein glucose-regulated protein 94 (GRP94) in liver tissues and GRP94 protein levels were compared through immunohistochemistry and Western blot assays., Results: TPN-soy animals had significantly higher serum total bilirubin, direct bilirubin, and γ-glutamyl transpeptidase and lower serum albumin than the controls (P < 0.01, each) or the TPN-FO group, which were similar to the controls (P < 0.01 cf. TPN). Damage to liver tissues of the TPN-FO group was much less than that of the TPN-soy group. GRP94 messenger RNA and protein levels in liver tissues of TPN-soy animals were significantly higher than that of the controls or TPN-FO rabbits, which were similar to the controls., Conclusions: Incorporating ω-3 fish oil in parenteral nutrition emulsion greatly prevented liver dysfunction and liver tissue damage in week-old rabbit kits, possibly by preventing endoplasmic reticulum stress.

Objective: To investigate the effect of peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist 15d-PGJ2 on the expressions of CXCL9, CXCL10 and CXCL11 in renal tubular epithelial cells (HK-2) stimulated by interferon-γ (IFN-γ) plus tumor necrosis factor-α (TNF-α)., Methods: HK-2 cells were stimulated with IFN-γ plus TNF-α only, or incubated with 15d-PGJ2 for 48 hours. CXCL9, CXCL10 and CXCL11 expressions were determined by real-time quantitative PCR (qRT-PCR) and ELISA., Results: IFN-γ plus TNF-α increased mRNA and protein expressions of CXCL9, CXCL10 and CXCL11 in HK-2 cells. PPAR-γ agonist 15d-PGJ2 inhibited the expressions of CXCL9, CXCL10 and CXCL11 in IFN-γ combined with TNF-α-induced HK-2 cells. Compared with the IFN-γ plus TNF-α group (untreated with 15d-PGJ2), the CXCL9, CXCL10 and CXCL11 were depressed by 76.8%, 78.7% and 81.9% at mRNA level and 66.9%, 86.6% and 39.9% at protein level in 2.0 ng/mL 15d-PGJ2-treated HK-2 cells, respectively (P<0.05)., Conclusion: PPAR-γ agonist 15d-PGJ2 could inhibit CXCL9, CXCL10 and CXCL11 production induced by IFN-γ combined with TNF-α in HK-2 cells.

Three forms of recombinant human keratinocyte growth factor 1 (rhKGF1) with or without the native signal peptide or a 23-amino acid truncation were expressed in Spodoptera frugiperda 9 (Sf9) cells by designing with insect codon usage. Immunoblotting demonstrated that these rhKGF1 proteins were recognized by a human anti-KGF1 antibody. The multiplicity of infection and timing of harvest had a significant effect on protein yield, protein quality, and cytotoxicity. Our results indicated that the native signal peptide directed KGF1 secretion from insect cells, reaching a maximum at 60 h postinfection. Although secretion of rhKGF1194 was less efficient than that of rhKGF1163 and rhKGF1140, protein secretion is an attractive pathway for simple purification of biologically active rhKGF1 at a high yield. Moreover, the sizes of rhKGF1194 and rhKGF1163 were similar (20 kDa), suggesting that the signal peptide may be recognized and removed in Sf9 cells. A 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay was used to analyze the biological function of rhKGF1, indicating that the three forms of rhKGF1 had a similar mitogenic function in BaF3 cells. Furthermore, to elucidate the effect of rhKGF1 on cytoprotection of liver cells, we used KGF1 pretreatment of an acute liver injury model. The results indicated that rhKGF1 prevented necrosis and apoptosis of CCl4-treated HL7702 cells in vitro and in vivo. These results suggest that KGF1 may be a candidate therapeutic drug for acute liver injury.

Avian pathogenic Escherichia coli (APEC) are responsible for heavy economic losses in poultry industry. Here we investigate DNA methylome of spleen and identify functional DNA methylation changes related to host response to APEC among groups of non-challenged chickens (NC), challenged with mild (MD) and severe pathology (SV). DNA methylation was enriched in the gene bodies and repeats. Promoter and CGIs are hypomethylated. Integration analysis revealed 22, 87, and 9 genes exhibiting inversely changed DNA methylation and gene expression in NC vs. MD, NC vs. SV, and MD vs. SV, respectively. IL8, IL2RB, and IL1RAPL1 were included. Gene network analysis suggested that besides inflammatory response, other networks and pathways such as organismal injury and abnormalities, cell signaling and molecular transport, are probably related to host response to APEC infection. Moreover, methylation changes in cell cycle processes might contribute to the lesion phenotype differences between MD and SV.

Age-related hearing loss - presbycusis - is the number one neurodegenerative disorder and top communication deficit of our aged population. Like many aging disorders of the nervous system, damage from free radicals linked to production of reactive oxygen and/or nitrogen species (ROS and RNS, respectively) may play key roles in disease progression. The efficacy of the antioxidant systems, e.g., glutathione and thioredoxin, is an important factor in pathophysiology of the aging nervous system. In this investigation, relations between the expression of antioxidant-related genes in the auditory portion of the inner ear - cochlea, and age-related hearing loss was explored for CBA/CaJ mice. Forty mice were classified into four groups according to age and degree of hearing loss. Cochlear mRNA samples were collected and cDNA generated. Using Affymetrix® GeneChip, the expressions of 56 antioxidant-related gene probes were analyzed to estimate the differences in gene expression between the four subject groups. The expression of Glutathione peroxidase 6, Gpx6; Thioredoxin reductase 1, Txnrd1; Isocitrate dehydrogenase 1, Idh1; and Heat shock protein 1, Hspb1; were significantly different, or showed large fold-change differences between subject groups. The Gpx6, Txnrd1 and Hspb1 gene expression changes were validated using qPCR. The Gpx6 gene was upregulated while the Txnrd1 gene was downregulated with age/hearing loss. The Hspb1 gene was found to be downregulated in middle-aged animals as well as those with mild presbycusis, whereas it was upregulated in those with severe presbycusis. These results facilitate development of future interventions to predict, prevent or slow down the progression of presbycusis.

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. The most common method for analyzing qRT-PCR data is to normalize mRNA levels of target genes to internal reference genes. Evaluating and selecting stable reference genes on a case-by-case basis is critical. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for normalization of mRNA expression in qRT-PCR analysis of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). For this purpose, three software tools (geNorm, NormFinder and BestKeeper) were used to investigate 10 candidate reference genes in nine developmental stages and five different tissues (epidermis, head, midgut, fat body and hemolymph) in three larval physiological stages (molting, feeding and wandering stages) of, S. exigua. With the exception of 18S ribosomal RNA (18S), all other candidate genes evaluated, β-actin1(ACT1), β-actin2 (ACT2), elongation factor1(EF1), elongation factor 2 (EF2), Glyceralde hyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), α-tubulin (TUB),proved to be acceptable reference genes. However, their suitability partly differed between physiological stages and different tissues. L10, EF2 and L17A ranked highest in all tissue sample sets. SOD, ACT2, GAPDH, EF1 and ACT1 were stably expressed in all developmental stage sample sets; ACT2, ACT1 and L10 for larvae sample sets; GAPDH, ACT1 and ACT2 for pupae and adults; SOD and L17A for males; and EF2 and SOD for females. The expression stability of genes varied in different conditions. The findings provided here demonstrated, with a few exceptions, the suitability of most of the 10 reference genes tested in tissues and life developmental stages. Overall, this study emphasizes the importance of validating reference genes for qRT-PCR analysis in S. exigua.

Objective: Coreceptors are important for HIV-1 entry into target cells and disease progression. The impact of HIV-1 and highly active antiretroviral treatment (HAART) on coreceptor expression has been little studied., Methods: Expression of C-C chemokine receptor (CCR) 5 and C-X-C chemokine receptor (CXCR) 4 on CD4+ and CD8 + T cells was compared in HIV-1-infected individuals who had/had not received HAART, and in healthy controls. Relationships between coreceptors and their chemokine ligands were studied., Results: This study included 23 controls and 88 HIV-1-infected individuals, 35 of whom were HAART naïve. Percentages of CCR5 and CXCR4+ CD8 + T cells were higher, and CXCR4+ CD4 + T cells were lower, in patients than in controls. Patients receiving HAART showed a higher percentage of CCR5 expression on CD4 + T cells compared with HAART-naïve patients. HIV-infected individuals had significantly increased levels of peripheral ligands for coreceptors, compared with controls; levels were significantly higher in those receiving HAART compared with the HAART-naïve., Conclusions: HIV-1 infection increases coreceptor expression on T cells; HAART increases CCR5 expression further and decreases CXCR4 expression, reversing the switch from CCR5 to CXCR4, which was significant for CD4 + T.

Ascorbate is a primary antioxidant and an essential enzyme cofactor in plants, which has an important effect on the development of plant root system. To investigate the molecular mechanisms of ascorbate accumulation during root development and reveal the key genes of the ascorbate biosynthesis and recycling pathways, the expression of 16 related genes together with ascorbate abundance were analyzed in the flesh and skin of radish (Raphanus sativus L.) fleshy root. The content of ascorbate decreased with root growth in both the flesh and skin. Expression of GDP-d-mannose pyrophosphorylase, GDP-d-mannose-3',5'-epimerase and d-galacturonate reductase were also decreased and correlated with ascorbate levels in the flesh. In the skin, the expression of GDP-d-mannose pyrophosphorylase and l-galactose dehydrogenase was correlated with ascorbate levels. These results suggested that ascorbate accumulation is affected mainly by biosynthesis rather than recycling in radish root, and the l-galactose pathway may be the major biosynthetic route of ascorbate, and moreover, the salvage pathway may also contribute to ascorbate accumulation. The data suggested that GDP-d-mannose pyrophosphorylase could play an important role in the regulation of ascorbate accumulation during radish fleshy taproot development., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Objective: To construct a eukaryotic expression plasmid harboring HBV fusion antigen gene, and to express it in 293T cells., Methods: The HBV fusion gene fragment was amplified by PCR from the plasmid pVAX1-HBV containing HBV fusion gene. After purified, the product was cloned into pMD18-T vector. The recombinant plasmid was confirmed by endonuclease digestion and sequencing analysis, and then subcloned into eukaryotic expression vector pIRES-neo. Then the recombinant expression plasmid pIRES-neo-HBAg was transfered into 293T cells by Lipofectamine(TM); 2000. The expression of HBV fusion antigen was identified by Western blotting, flow cytometry and immunofluorescence cytochemistry., Results: The eukaryotic expression vector pIRES-neo-HBAg was constructed successfully. The expression of fusion antigen could be detected in the pIRES-neo-HBAg transfected 293T cells by Western blotting, immunofluorescence cytochemistry and flow cytometry., Conclusion: The eukaryotic expression plasmid pIRES-neo-HBAg is successfully constructed and the fusion antigen is expressed in 293T cells.

Currently the treatment of Mycobacterium tuberculosis (TB) infection is largely limited due to the prevalence of multidrug resistance strains. Over-expressing the efflux pumps such as the ATP-binding cassette (ABC) transporter has been reported to significantly contribute to its resistance to several antibiotics. This study investigated the expression profile of one important ABC efflux pump, Rv1217c-Rv1218c, by quantitative real-time PCR (RT-qPCR) in clinical isolates from China, which also revealed its association with the multidrug resistance of M. tuberculosis. Significantly increased expressions of Rv1217c and Rv1218c at transcriptional level have been observed in multidrug-resistant TB group (MDR-TB) compared to those of the drug-susceptible group (P < 0.05), when H37Rv strain was used as the control. Furthermore, correlation analysis revealed that the over-expression of both Rv1217c and Rv1218c resulted in the higher minimum inhibition concentrations (MICs) of rifampicin (RIF) (OR = 1.01, P < 0.05 of Rv1217c; OR = 1.23, P < 0.05 of Rv1218c), while the over-expression of Rv1218c only led to the higher MICs of isoniazid (INH) (OR = 1.17, P < 0.05). Our findings contributed to the better understanding of the molecular mechanisms of ABC efflux pumps, in particular Rv1217c-Rv1218c, in M. tuberculosis and will assist in developing new antibiotic treatments for multidrug-resistant M. tuberculosis in the future.

Real-time quantitative reverse transcription PCR (RT-qPCR) is a rapid and reliable method for gene expression studies. Normalization based on reference genes can increase the reliability of this technique; however, recent studies have shown that almost no single reference gene is universal for all possible experimental conditions. In this study, eight frequently used reference genes were investigated, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin2/7 (ACT), Tubulin alpha-5 (TUA), Tubulin beta-1 (TUB), 18S ribosomal RNA (18SrRNA), RNA polymerase-II transcription factor (RPII), Elongation factor 1-b (EF-1b) and Translation elongation factor 2 (TEF2). Expression stability of candidate reference genes was examined across 27 radish samples, representing a range of tissue types, cultivars, photoperiodic and vernalization treatments, and developmental stages. The eight genes in these sample pools displayed a wide range of Ct values and were variably expressed. Two statistical software packages, geNorm and NormFinder showed that TEF2, RPII and ACT appeared to be relatively stable and therefore the most suitable for use as reference genes. These results facilitate selection of desirable reference genes for accurate gene expression studies in radish., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recent studies showed A20 inactivation by deletion, mutation and promoter methylation in ocular adnexal mucosa-associated lymphoid tissue lymphoma. However, the incidences of A20 abnormalities and their clinical impact remain for the most part unknown. It is also unknown whether ABIN-1 and ABIN-2, the components of the A20 NF-κB inhibitor complex, are inactivated by genetic changes in ocular adnexal mucosa-associated lymphoid tissue lymphoma. A total of 105 cases were investigated for A20 mutation/deletion, ABIN-1/2 mutation, MALT1 and IGH involved translocation. Somatic mutation was seen frequently in A20 (28.6%) but rarely in ABIN-1 (1%) and ABIN-2 (1%). A20 mutations were significantly associated with A20 heterozygous deletion, and both were mutually exclusive from the MALT1 or IGH involved translocations. A20 mutation/deletion was also significantly associated with increased expression of the NF-κB target genes CCR2, TLR6 and BCL2. The cases with A20 mutation/deletion required significantly higher radiation dosages to achieve complete remission than those without these abnormalities.

An esterase PE10 (279 aa) from Pelagibacterium halotolerans B2(T) was cloned and overexpressed in Escherichia coli Rosetta in a soluble form. The deduced protein was 29.91 kDa and the phylogenetic analysis of the deduced amino acids sequence showed it represented a new family of lipolytic enzymes. The recombinant protein was purified by Ni-NTA affinity chromatography column and the characterization showed its optimal temperature and pH were 45 °C and pH 7.5, respectively. Substrate specificity study showed PE10 preferred short chain p-nitrophenyl esters and exhibited maximum activity toward p-nitrophenyl acetate. In addition, PE10 was a halotolerant esterase as it was still active under 4 M NaCl. Three-dimensional modeling of PE10 suggested that the high negative electrostatic potential on the surface may relevant to its tolerance to high salt environment. With this halotolerance property, PE10 could be a candidate for industrial use.

In the present study, the potential of RNA interference (RNAi) as a gene silencing tool and the resultant effects on Ascaris suum larval development was examined by targeting a gene (represented by the EST 06G09) specifically expressed in the infective larvae of A. suum. BALB/c mice were infected with RNAi-treated larvae. The results showed that the target gene was silenced after soaking for 72 h, and the survival rate of the RNAi-treated larvae was reduced by 17.25% (P<0.01). A significant difference (P<0.05) was detected in the numbers of larvae collected from the livers and lungs of infected mice 4 days after infection with untreated larvae (164.29 ± 21.51) and RNAi-treated larvae (71.43 ± 14.35). Significant differences (P<0.01) were also found in the body length and width between untreated larvae (480 ± 105.77 μm for length and 23.93 ± 3.72 μm for width) and RNAi-treated larvae (400.57 ± 71.31 μm for length and 20.20 ± 2.43 μm for width). These results show that the gene represented by EST 06G09 may play a role in the development of A. suum larvae.

The objective of this study was to compare the differences of gene expression profiles between intramuscular and subcutaneous adipocytes originated from the isolated preadipocytes in vitro. Cytosolic triglyceride determination indicated that subcutaneous adipocytes accumulated more lipid than intramuscular adipocytes did at the late stage of differentiation. Microarray assay revealed that 172 probes representing 133 genes were differentially expressed, among which 46 genes were highly expressed in intramuscular adipocytes and the other 87 genes were highly expressed in subcutaneous adipocytes. Real-time PCR confirmed that genes related to lipid metabolism, such as LPL, FABP4, FABP5 and OSBPL10, were predominantly expressed in subcutaneous adipocytes, whereas BMP4 and BMP7 were highly expressed in intramuscular adipocytes. The results indicated that the accumulation of lipid mass in subcutaneous adipocytes might be due to the highly expressed genes related to lipid metabolism, and the high levels of BMP4 and BMP7 in intramuscular adipocytes suggested that BMPs might be involved in the differentiation of intramuscular adipocytes., (Copyright (c) 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.)

Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, plays a key role in cardiac dysfunction in sepsis. Low circulating levels of insulin-like growth factor 1 (IGF-1) are found in sepsis, although the influence of IGF-1 on septic cardiac defect is unknown. This study was designed to examine the impact of IGF-1 on LPS-induced cardiac contractile and intracellular Ca2+ dysfunction, activation of stress signal and endoplasmic reticulum (ER) stress. Mechanical and intracellular Ca2+ properties were examined in cardiomyocytes from Fast Violet B and cardiac-specific IGF-1 overexpression mice treated with or without LPS (4 mg kg(-1), 6 h). Reactive oxygen species (ROS), protein carbonyl formation and apoptosis were measured. Activation of mitogen-activated protein kinase pathways (p38, c-jun N-terminal kinase [JNK] and extracellular signal-related kinase [ERK]), ER stress and apoptotic markers were evaluated using Western blot analysis. Our results revealed decreased peak shortening and maximal velocity of shortening/relengthening and prolonged duration of relengthening in LPS-treated Fast Violet B cardiomyocytes associated with reduced intracellular Ca2+ decay. Accumulation of ROS protein carbonyl and apoptosis were elevated after LPS treatment. Western blot analysis revealed activated p38 and JNK, up-regulated Bax, and the ER stress markers GRP78 and Gadd153 in LPS-treated mouse hearts without any change in ERK and Bcl-2. Total protein expression of p38, JNK, and ERK was unaffected by either LPS or IGF-1. Interestingly, these LPS-induced changes in mechanical and intracellular Ca2+ properties, ROS, protein carbonyl, apoptosis, stress signal activation, and ER stress markers were effectively ablated by IGF-1. In vitro LPS exposure (1 microg mL(-1)) produced cardiomyocyte mechanical dysfunction reminiscent of the in vivo setting, which was alleviated by exogenous IGF-1 (50 nM). These data collectively suggested a beneficial of IGF-1 in the management of cardiac dysfunction under sepsis.

Avian pathogenic Escherichia coli (APEC) and uropathogenic E. coli (UPEC) establish infections in extraintestinal habitats of different hosts. As the diversity, epidemiological sources and evolutionary origins of extraintestinal pathogenic E. coli (ExPEC) are so far only partially defined, in the present study,100 APEC isolates and 202 UPEC isolates were compared by their content of virulence genes and phylogenetic groups. The two groups showed substantial overlap in terms of their serogroups, phylogenetic groups and virulence genotypes, including their possession of certain genes associated with large transmissible plasmids of APEC. In a chicken challenge model, both UPEC U17 and APEC E058 had similar LD(50), demonstrating that UPEC U17 had the potential to cause significant disease in poultry. To gain further information about the similarities between UPEC and APEC, the in vivo expression of 152 specific genes of UPEC U17 and APEC E058 in both a murine urinary tract infection (UTI) model and a chicken challenge model was compared with that of these strains grown statically to exponential phase in rich medium. It was found that in the same model (murine UTI or chicken challenge), various genes of UPEC U17 and APEC E058 showed a similar tendency of expression. Several iron-related genes were upregulated in the UTI model and/or chicken challenge model, indicating that iron acquisition is important for E. coli to survive in blood or the urinary tract. Based on these results, the potential for APEC to act as human UPEC or as a reservoir of virulence genes for UPEC should be considered. Further, this study compared the transcriptional profile of virulence genes among APEC and UPEC in vivo.

We investigated laminin beta1 expression in the hippocampi of patients with intractable epilepsy and explored the role of laminin beta1 in the pathogenesis of this condition. Fluorescence quantitative PCR, immunofluorescence, immunohistochemistry and Western blotting were used to measure laminin beta1 expression in surgically removed hippocampi of patients with intractable epilepsy, and the results were compared with control hippocampi. Fluorescence quantitative PCR showed increased expression of laminin beta1 mRNA in patient hippocampi compared with control tissues. Immunohistochemical staining demonstrated that laminin beta1 protein expression was significantly increased in patient hippocampi, and immunofluorescence microscopy showed accumulation of laminin beta1 in the cell membrane and cytoplasm of patient hippocampi. These findings were confirmed by Western blotting of protein preparations from patient hippocampi. Elevated expression of laminin beta1 mRNA and protein in the hippocampus suggests that laminin beta1 may play a role in the development of epileptic seizures in patients with intractable epilepsy.

Unlabelled: Presbycusis - age-related hearing loss - is the number one communicative disorder and one of the top three chronic medical condition of our aged population. High-throughput technologies potentially can be used to identify differentially expressed genes that may be better diagnostic and therapeutic targets for sensory and neural disorders. Here we analyzed gene expression for a set of GABA receptors in the cochlea of aging CBA mice using the Affymetrix GeneChip MOE430A. Functional phenotypic hearing measures were made, including auditory brainstem response (ABR) thresholds and distortion-product otoacoustic emission (DPOAE) amplitudes (four age groups). Four specific criteria were used to assess gene expression changes from RMA normalized microarray data (40 replicates). Linear regression models were used to fit the neurophysiological hearing measurements to probe-set expression profiles. These data were first subjected to one-way ANOVA, and then linear regression was performed. In addition, the log signal ratio was converted to fold change, and selected gene expression changes were confirmed by relative real-time PCR., Major Findings: expression of GABA-A receptor subunit alpha6 was upregulated with age and hearing loss, whereas subunit alpha1 was repressed. In addition, GABA-A receptor associated protein like-1 and GABA-A receptor associated protein like-2 were strongly downregulated with age and hearing impairment. Lastly, gene expression measures were correlated with pathway/network relationships relevant to the inner ear using Pathway Architect, to identify key pathways consistent with the gene expression changes observed.

Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, approximately 1.4 kb 3' of the promoter, but inactive approximately 6 kb 3', indicating that the activity of the core enhancer was distance-dependent. A longer segment--the core enhancer plus approximately 1 kb of 3' flanking material, including the 3' matrix attachment region--was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3' flank includes binding sites for at least two activators.

Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, has been widely suggested to play a role in the pathogenesis of Alzheimer's disease. Supporting this, levels of TGF-beta are elevated in the cerebrospinal fluid, sera, and brain of patients with Alzheimer's disease. Since TGF-beta is neuroprotective, whereas Alzheimer's disease is typified by neurodegeneration, we speculated that defects in TGF-beta signaling might abrogate its neuroprotective properties. Consistently with an increase in TGF-beta in Alzheimer's disease, we found significant increases in phospho-Smad2, a major downstream signaling molecule of TGF-beta, in hippocampal neurons of Alzheimer's disease compared with age-matched control patients. However, in contrast to an expected nuclear localization, phosphorylated Smad2 in Alzheimer's disease was predominantly, and ectopically, found in the neuronal cytoplasm, specifically colocalized with neurofibrillary tangles and granulovacuolar degeneration. Given that a nuclear localization is required to regulate the transcription of TGF-beta target genes to afford neuroprotection, the ectopic localization of phosphorylated Smad2 suggests a defect in the Smad-mediated signaling pathway of TGF-beta in Alzheimer's disease and consequent loss of neuroprotective function.

To characterize how signaling by TLR ligands can be modulated by non-TLR ligands, murine RAW 264.7 cells were treated with LPS, IFN-gamma, 2-methyl-thio-ATP (2MA), PGE(2), and isoproterenol (ISO). Ligands were applied individually and in combination with LPS, for 1, 2, and 4 h, and transcriptional changes were measured using customized oligo arrays. We used nonadditive transcriptional responses to dual ligands (responses that were reproducibly greater or less than the expected additive responses) as a measure of pathway interaction. Our analysis suggests that cross-talk is limited; <24% of the features with significant responses to the single ligands responded nonadditively to a dual ligand pair. PGE(2) and ISO mainly attenuated, while 2MA enhanced, LPS-induced transcriptional changes. IFN-gamma and LPS cross-regulated the transcriptional response induced by each other: while LPS preferentially enhanced IFN-gamma-induced changes in gene expression at 1 h, IFN-gamma signaling primarily attenuated LPS-induced changes at 4 h. Our data suggest specific cross-talk mechanisms: 1) LPS enhances the expression of IFN-gamma-response genes by augmenting STAT1 activity and by activating NF-kappaB, which synergizes with IFN-gamma-induced transcriptional factors; 2) IFN-gamma attenuates the late LPS transcriptional response by increasing the expression of suppressor of cytokine signaling 1 and cytokine-inducible SH2-containing protein expression; 3) 2MA modulates LPS secondary transcriptional response by increasing IFN-beta and inhibiting IL-10 gene expression; 4) PGE(2) and ISO similarly regulate the LPS transcriptional response. They increase IL-10 transcription, resulting in attenuated expression of known IL-10-suppressed genes.

Background: As phenotypic features derived from heritable characters, the topologies of metabolic pathways contain both phylogenetic and phenetic components. In the post-genomic era, it is possible to measure the "phylophenetic" contents of different pathways topologies from a global perspective., Results: We reconstructed phylophenetic trees for all available metabolic pathways based on topological similarities, and compared them to the corresponding 16S rRNA-based trees. Similarity values for each pair of trees ranged from 0.044 to 0.297. Using the quartet method, single pathways trees were merged into a comprehensive tree containing information from a large part of the entire metabolic networks. This tree showed considerably higher similarity (0.386) to the corresponding 16S rRNA-based tree than any tree based on a single pathway, but was, on the other hand, sufficiently distinct to preserve unique phylogenetic information not reflected by the 16S rRNA tree., Conclusion: We observed that the topology of different metabolic pathways provided different phylogenetic and phenetic information, depicting the compromise between phylogenetic information and varying evolutionary pressures forming metabolic pathway topologies in different organisms. The phylogenetic information content of the comprehensive tree is substantially higher than that of any tree based on a single pathway, which also gave clues to constraints working on the topology of the global metabolic networks, information that is only partly reflected by the topologies of individual metabolic pathways.

Objective: To study the effect of diamide on the expression of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in cultured human umbilical vein endothelial cells., Methods: After exposure of the endothelial cells (ECs) to different concentrations of diamide for 4 hours, the MIP-1 alpha mRNA in the cells was detected by nuclease S1 protection assay and the MIP-1 alpha protein in those cells was determined by cell enzyme-linked immunosorbent assay. The chemotactic activity of MIP-1 alpha in the conditioned medium of ECs treated with diamide for peripheral blood monocytes was tested by microfilter method using modified Boyden chambers., Results: Incubation of ECs with 5 micro mol/L diamide resulted in a 2.4-fold increase in the level of MIP-1 alpha mRNA expression as compared with the control group (t = 8.70, P < 0.05). Exposure of ECs to 1 micro mol/L, 5 micro mol/L and 10 micro mol/L diamide resulted in a 0.9-fold, 1.2-fold, and 0.7-fold increase in the level of MIP-1 alpha protein expression respectively, as compared with the control group (F = 35.65, P < 0.05). Chemotactic assay showed that the migration distance of monocytes towards the conditioned medium (CM) of ECs treated with 5 micromol/L diamide was 99.50 microm +/- 4.31 microm, which was significantly more than the 66.47 microm +/- 3.25 microm towards the conditioned medium of ECs in the non-diamide group, the chemokinetic group (67.03 microm +/- 6.83 microm) and the random migration group (65.40 microm +/- 3.36 microm) (F = 404.31, P < 0.05). The results revealed that there might be chemotactic substances in the conditioned medium of 5 micro mol/L diamide treated ECs. The migration distance of monocytes towards the conditioned medium of the ECs exposed to 5 micromol/L diamide was significantly reduced to 82.80 microm +/- 6.88 microm after the addition of goat anti-human MIP-1 alpha antibody (F = 192.25, P < 0.05), which indicates the chemotactic activity of MIP-1 alpha in the conditioned medium of the ECs in the diamide group., Conclusions: Diamide, a lipid peroxidation inducer, could stimulate ECs to produce high levels of MIP-1 alpha with chemotactic activity, and may play an important role in atherogenesis through attraction of peripheral blood monocytes into arterial intima.

Objective: To investigate the role of substance P in the formation of hypertrophic scar., Methods: Dermal fibroblasts derived from human normal skin were cultured with substance P alone or together with selective non-peptide NK-1 tachykinin antagonist, L-703, 606 oxalate salt. The effect of substance P on proliferation of fibroblasts was measured by MTT assay. Furthermore, the TGF-beta 1 mRNA expression in the fibroblasts was determined by in situ hybridization and image analysis., Results: Substance P enhanced fibroblast proliferation dose-dependently, which showed the maximum rate when the concentration of substance P was 25 ng/ml or higher in the culture media. By 48 hours cultured with 25 ng/ml of substance P, the fibroblasts expressed TGF-beta 1 mRNA more significantly than the fibroblasts without substance P. The effects of substance P on both fibroblast proliferation and TGF-beta 1 mRNA expression could be antagonized by L-703, 606 oxalate salt., Conclusion: The results suggest that substance P may play an important role in phenotype changes of fibroblasts in skin scarring.

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis. Recent reports suggest that OPG may function as a protector of arterial calcification and survival of endothelial cells. However, the role and expression of OPG in vascular wall is unclear. Here we report that OPG was highly expressed in vascular smooth muscle cells (VSMC) but not in endothelial cells. Platelet-derived growth factor (PDGF), basic fibroblast growth factor, angiotensin II, tumor necrosis factor alpha and interleukin-1beta upregulated OPG expression in VSMC. Moreover, inhibition of phosphatidylinositol 3-kinase/Akt or P38-signal pathway abrogated PDGF-induced OPG expression. Our results suggest that OPG may be an important determinant of vascular homeostasis.

The primary mesenchyme cells (PMCs) of the sea urchin embryo have been an important model system for the analysis of cell behavior during gastrulation. To gain an improved understanding of the molecular basis of PMC behavior, a set of 8293 expressed sequenced tags (ESTs) was derived from an enriched population of mid-gastrula stage PMCs. These ESTs represented approximately 1200 distinct proteins, or about 15% of the mRNAs expressed by the gastrula stage embryo. 655 proteins were similar (P<10(-7) by BLAST comparisons) to other proteins in GenBank, for which some information is available concerning expression and/or function. Another 116 were similar to ESTs identified in other organisms, but not further characterized. We conservatively estimate that sequences encoding at least 435 additional proteins were included in the pool of ESTs that did not yield matches by BLAST analysis. The collection of newly identified proteins includes many candidate regulators of primary mesenchyme morphogenesis, including PMC-specific extracellular matrix proteins, cell surface proteins, spicule matrix proteins and transcription factors. This work provides a basis for linking specific molecular changes to specific cell behaviors during gastrulation. Our analysis has also led to the cloning of several key components of signaling pathways that play crucial roles in early sea urchin development.

Myostatin, a TGF-beta family member, is a negative regulator of muscle growth. Here, we generated transgenic mice that expressed myostatin mutated at its cleavage site under the control of a muscle specific promoter creating a dominant negative myostatin. These mice exhibited a significant (20-35%) increase in muscle mass that resulted from myofiber hypertrophy and not from myofiber hyperplasia. We also evaluated the role of myostatin in muscle degenerative states, such as muscular dystrophy, and found significant downregulation of myostatin. Thus, further inhibition of myostatin may permit increased muscle growth in muscle degenerative disorders.

Liposome encapsulated haemoglobin (LEH), a blood substitute, has been shown to increase serum IL-6 levels in mice, but the cellular source of this cytokine is unknown. These experiments were conducted to determine cytokine gene expression by macrophages (P388D1) and endothelial cells (EOMA) after LEH treatment in vitro. P388D1 and EOMA cells were incubated with LEH, final concentration of 10%, for 0, 0.5, 1, 2, 4, 8 and 24 h. Total RNA was extracted at the above times and semi-quantitative RT-PCR was carried out to detect cytokine mRNA expression in the presence of competitive internal standards specific for mouse IL-1 beta, IL-6, TGF-beta, TNF-alpha and GM-CSF. The results demonstrated that although constitutive cytokine gene expression exists in both cell lines, the level of IL-6 mRNA was prominently elevated by incubation with LEH, rapidly reaching a peak at about 4 h and gradually declining over the next 20 h after LEH treatment. In contrast, there was no significant change in mRNA accumulation of IL-1 beta, TGF-beta, TNF-alpha or GM-CSF at all times. These in vitro findings suggest that macrophages and endothelial cells may be the cellular sources of the elevated levels of IL-6 found in serum after in vivo LEH administration.

To investigate substrates of recognition memory, the cellular expression of Fos protein in rat brain has been studied after groups of rats were either shown sets of novel or highly familiar objects, or were exposed to the same pattern of illumination without objects being shown. Counts of stained nuclei were made in eight brain regions, where information about novel or familiar visual stimuli is likely to be processed or stored. The counts were relatively high in occipital visual association cortex and area TE of temporal cortex, intermediate in perirhinal cortex, entorhinal cortex, anterior cingulate cortex and the diagonal band of Broca, and low in the hippocampal formation and mediodorsal nucleus of the thalamus. The number of Fos-stained cells was significantly higher for the rats shown novel objects than for those shown familiar objects in perirhinal cortex, area TE, occipital cortex and anterior cingulate cortex. Arguments are advanced that these differences in counts indicate areas involved in the processing and/or storage of information about the novelty or familiarity of visual stimuli, information important to recognition memory.

This paper describes the use of both mammalian and bacterial expression systems as tools to study the structural and functional relationships of proteins encoded by cDNAs to both rat and human aryl sulfotransferases. In particular, we describe the use of the mammalian COS cell system for functional expression studies, and the use of Escherichia coli for the expression and purification of a sulfotransferase fusion protein suitable as an antigen for the generation of sulfotransferase antibodies.